Most reported FRET indicators are based on historically first (BFP)CFP/YFP pairs [12–15]. However, these FRET pairs are not really the most convenient and effective. Indeed, spectral separation of overlapping cyan and yellow emission spectra can never be complete, and use of narrow band-pass filters results in dramatic loss of emission. Besides, the relative high levels of autofluorescence in blue-cyan region of visible spectrum and phototoxicity using near-UV excitation further complicates their application.
Reported FRET pairs containing red fluorescent acceptors suffer from tetramerization and demonstrate lower contrast [16, 17]. The closest competitor of CaspeR3 is a high contrasting caspase-3 indicator MiCy-mKO , However, it was observed that mKO converts from orange to green fluorescent form upon blue light illumination , that can hamper interpretation of FRET changes in this and other mKO-based techniques.
The high extinction coefficient of TagRFP makes it a preferable FRET acceptor for the green fluorescent proteins. It is of note that the actual FRET efficiency is an inverse 6th power distance dependency, leading to a quick drop of the detected FRET efficiency at donor-acceptor proximities above R0. For instance, under similar conditions, we expect that, given the Förster radii 5.28 nm and 5.74 nm for TagGFP-mCherry and TagGFP-TagRFP respectively, the latter would display 1.4-fold more FRET than a sensor comprised of the TagGFP-mCherry pair.
The superior Förster radius is caused by an 1.5-fold increased spectral overlap for the TagGFP-TagRFP pair as compared to the TagGFP-mCherry pair. While not contributing to the FRET efficiency, the significantly increased quantum yield of TagRFP is highly beneficial for acceptor-based ratiometric FRET studies.
Altogether, the combined advantages of the TagGFP-TagRFP make it the FRET pair of choice both for the ratiometric FRET analyses and FLIM assays to monitor interaction of proteins of interest in living cells, as well as to generate high contrast FRET-based genetically encoded sensors for various analites and protein activities.